The family Flaviviridae includes over 60 human pathogens, including hemorrhagic fever viruses [e.g. dengue, yellow fever (YF)] and encephalitis viruses [e.g. West Nile virus, Japanese encephalitis virus (JE)]. These viruses have a high degree of homology and immunologic cross-reactivity. The long-term objective of this project is to determine the effect of flavivirus-specific CD4+ and CD8+ T cell responses induced by primary flavivirus infection on the immune response to subsequent infection with other flaviviruses. Sequential flavivirus infections occur in nature and there are examples of cross-protection as well as enhanced disease in secondary flavivirus infections. In Aim 1, we plan to identify novel flavivirus-specific CD4+ and CD8+ T cell epitopes. We will then characterize effector responses (cytokine production, proliferation) to homologous and heterologous viruses. In Aim 2, we will use two closely related viruses to model the immunologic effects of sequential flavivirus infection: yellow fever (YF)17D and a candidate Japanese encephalitis virus (JE) vaccine comprised of a live attenuated chimeric YF that contains the pre-membrane (prM) and envelope (E) regions of a heterologous flavivirus, JE. We will quantitate the epitope-specific T cell responses in primary and secondary flavivirus infection, and determine whether secondary infections alter the T cell epitope hierarchy to memory/recall (nonstructural) or novel/naive (structural) antigens. In Aim 3, we will examine associations during primary and secondary immunization with YF17D and chimeric YF/JE between flavivirus-specific T cell responses and viremia and antibody responses. These studies will help to elucidate beneficial and detrimental aspects of heterologous viral immunity and will help in the development of novel vaccines against these biological threats.